To fully understand how neurons interact with each other to generate network function, it is necessary to obtain information about the activity of the entire population of neurons in the system. Toward this goal, we combine 4-D calcium imaging (with spinning-disc confocal microscopy and a high-speed Piezo device) which enables monitoring of the activity of neurons in a 3-dimension space in real time, with automated cell sorting which enables extracting the activity of thousands of neurons from the recording. We previously reported on the experimental system and the method of cell sorting. In this presentation, we introduce a "standard ventral nerve cord (VNC)", which allows statistical analyses across different preparations.
We used the genetically encoded calcium indicator GCaMP6 to detect the calcium signals of the cells. We crossed the UAS-GCaMP6 transgenic line to elav-Gal4 to express GCaMP6 in all neurons, isolated the VNC from the first instar larvae, and recorded the activities of the neurons within the entire VNC, which occur spontaneously and reflect fictive forward/backward locomotion and other behavioral states. We then used custom-made cell sorting methods to automatically extract the activity pattern of thousands of neurons. Since the position of the neurons vary from an individual to another, to compare the activity patterns between different larvae, it is necessary to assign the activity patterns to a standard VNC. We therefore created a standard VNC using symmetric diffeomorphic registration on the sample images in a multi-resolution approach. The creation of the standard brain VNC now allows statistical analyses of the activity data obtained from multiple larvae. We are now trying to combine these experimental and data-analyses methods with pharmacology to study the effect of neuromodulators on the dynamics of the neural network.